CAJ | 학술논문

为揭示 Toll 样受体家族基因 TLR9在鲫(Carassius auratus L.,缩写为 Ca)免疫过程中的作用,本研究利用RT-PCR和RACE技术克隆获得了鲫TLR9(CaTLR9)基因cDNA,并就注射外源性人绒毛膜促性激素(hCG)和不同性腺周期对该基因在鲫肠道表达的影响进行了分析。结果表明, CaTLR9 cDNA全长3509 bp,包含3195 bp的开放阅读框(ORF)、72 bp的5′非编码区和242 bp的3′非编码区。ORF编码1064个氨基酸组成的蛋白质。该蛋白包含19个氨基酸组成的信号肽,15个富含亮氨酸的重复结构域(LRR),1个跨膜区和1个TIR结构域。系统进化分析表明, CaTLR9与其他鲤科鱼类同源性较高,与鲤(Cyprinus carpio)相似度为92%,其次是草鱼(Ctenopharyngodon idella)和斑马鱼(Danio rerio),分别为83%和79%。实时荧光定量PCR结果表明, CaTLR9在鲫脾中表达量最高,其次为肾和鳃,其他组织的表达量均较低。鲫肠道 TLR9在非繁殖期的表达水平明显低于繁殖期,注射适当剂量的外源 hCG 可显著上调鲫肠道TLR9的表达。本研究对揭示鲫非特异性免疫能力调节机制,提高鲫健康养殖水平具有一定参考价值。
위게시 Toll 양수체가족기인 TLR9재즉(Carassius auratus L.,축사위 Ca)면역과정중적작용,본연구이용RT-PCR화RACE기술극륭획득료즉TLR9(CaTLR9)기인cDNA,병취주사외원성인융모막촉성격소(hCG)화불동성선주기대해기인재즉장도표체적영향진행료분석。결과표명, CaTLR9 cDNA전장3509 bp,포함3195 bp적개방열독광(ORF)、72 bp적5′비편마구화242 bp적3′비편마구。ORF편마1064개안기산조성적단백질。해단백포함19개안기산조성적신호태,15개부함량안산적중복결구역(LRR),1개과막구화1개TIR결구역。계통진화분석표명, CaTLR9여기타리과어류동원성교고,여리(Cyprinus carpio)상사도위92%,기차시초어(Ctenopharyngodon idella)화반마어(Danio rerio),분별위83%화79%。실시형광정량PCR결과표명, CaTLR9재즉비중표체량최고,기차위신화새,기타조직적표체량균교저。즉장도 TLR9재비번식기적표체수평명현저우번식기,주사괄당제량적외원 hCG 가현저상조즉장도TLR9적표체。본연구대게시즉비특이성면역능력조절궤제,제고즉건강양식수평구유일정삼고개치。
TLR9, a member of the Toll-like receptor family, plays a key role in the innate immune system by identify-ing the DNA CpG motif of bacteria and viruses. Using rapid amplification of cDNA ends (RACE), we cloned the full length TLR9 cDNA sequence of crucian carp (Carassius auratus L., Abbr. Ca). We then evaluated the effects of ex-ogenous hCG and gonad development on CaTLR9 expression using real-time PCR. The CaTLR9 cDNA was 3 509 bp in length with a 3′-untranslated region (242 bp), a 5′-untranslated region (72 bp), and a 3 195 bp open reading frame (ORF). The ORF of CaTLR9 cDNA encodes a 1 064 amino acid protein with a signal peptide of 19 amino acids, 15 leucine rich repeat (LRR) motifs, one transmembrane zone, and one Toll/IL-1 receptor (TIR) domain. The amino acid sequence of CaTLR9 possessed 92%, 83%and 79%identity with the TLR9s of Cyprinus carpio, Ctenopharyngodon idella and Danio rerio, respectively. The expression level of TLR9 was highest in the spleen of crucian carp, followed by the kidney and gill. The level of expression was low in the remaining tissues. The expression of TLR9 was higher during the breeding season than the non-breeding season. Additionally, injections of exogenous hCG significantly up-regulated the expression of TLR9 in the intestine of crucian carp. Our results suggest that TLR9 is a new candidate target gene for investigating non-specific immunity in crucian carp and improving the health of cultured fish.